8-Nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP) is formed endogenously under conditions of excess production of NO coupled with oxidative stress. Recently, we found that the electrophilic nitro moiety underwent nucleophilic substitution with hydrogen sulfide anion (HS − ) to yield the novel product 8-SH-cGMP. Endogenous 8-SH-cGMP levels are yet to be identified, however. In this study, we precisely quantified endogenous formation of 8-SH-cGMP in various tissues and cells by means of liquid chromatography- electrospray ionization (ESI)-tandem mass spectrometry (LC–ESI–MS/MS). Mouse tissue homogenates as well as lysates of various mammalian cultured cells were subjected to LC–ESI–MS/MS analysis with a Thermo TSQ vantage triplequadrupole mass spectrometer equipped with a reverse-phase HPLC column. For precise quantification, the stable isotope dilution method was employed: recovery efficiency of the analyte was corrected based on the recovery of the stable isotope-labeled derivative (8-SH-[ 13 C 10 ]cGMP) spiked into the extracts. This method warrants highly sensitive and specific identification of 8-SH-cGMP: detection limit, >1pmol/ml. According to the LC–MS/MS analyses with stable isotope dilution method, we could quantify the formation of 8-SH-cGMP in mouse tissues and various cells in culture. The highest concentration of 8-SH-cGMP determined in cells was over 10μM. This sulfhydryl derivative of cGMP is also shown to have a protein kinase G-activating potential with strong resistance to phosphodiesterase-catalyzed degradation. The significant endogenous formation of 8-SH-cGMP therefore may suggest its physiological function as a unique second messenger for hydrogen sulfide.